Reversible NaCl-induced aggregation of a monoclonal antibody at low pH: Characterization of aggregates and factors affecting aggregation

• Published in: European journal of pharmaceutics and biopharmaceutics 2016-10, Vol.107, p.310-320
• Main Authors: Bickel, Fabian, Herold, Eva Maria, Signes, Alba, Romeijn, Stefan, Jiskoot, Wim, Kiefer, Hans
• Format: Article
• Language: English
• Subjects: Acidic conditions; Aggregate; Antibodies, Monoclonal - chemistry; Chromatography, Gel; Chromatography, High Pressure Liquid; Circular Dichroism; Downstream processing; Hydrogen-Ion Concentration; IgG; MAb; Nephelometry and Turbidimetry; Osmolar Concentration; Particle Size; Reversibility; Salt; Sodium Chloride - chemistry; Spectroscopy, Fourier Transform Infrared; Trehalose - chemistry
• ISSN: 0939-6411; 1873-3441
• DOI: 10.1016/j.ejpb.2016.07.020

[Display omitted] We investigated the influence of pH and sodium chloride concentration on aggregation kinetics of a monoclonal antibody. Aggregation was induced by sodium chloride addition at low pH. Protein conformation before and after salt addition was determined as well as the reversibility of aggregation. Aggregation was monitored at pH values between 2 and 7 with NaCl up to 1.5M by turbidity measurement and size-exclusion chromatography. Particle size distribution was assessed by using size-exclusion chromatography as well as nanoparticle tracking analysis and flow imaging microscopy. Structural changes were monitored by circular dichroism, Fourier transform infrared and fluorescence spectroscopy. Thermal stability was measured by differential scanning fluorimetry. Aggregation propensity was maximal at low pH and high ionic strength. While thermal stability decreased with pH, the secondary structure remained unchanged down to pH 3.5 and up to 1.5M NaCl. Precipitated protein could be largely reverted to monomers by dilution into salt-free buffer. The re-solubilized antibody was indistinguishable in structure, solubility and monodispersity from the unstressed protein. Also, binding to Protein A was steady. Aggregation could be reduced in the presence of trehalose. The results suggest a reversible aggregation mechanism characterized by a limited change in tertiary structure at low pH and a subsequent loss of colloidal stability resulting from electrostatic repulsion once salt is added to the sample. The experimental setup is robust and allows high-throughput quantification of the effect of additives on aggregation kinetics.